Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
  • A61F2/02—Prostheses implantable into the body
  • A61F2/04—Hollow or tubular parts of organs, e.g. bladders, tracheae, bronchi or bile ducts
  • A61F2/06—Blood vessels
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2250/00—Special features of prostheses classified in groups A61F2/00 - A61F2/26 or A61F2/82 or A61F9/00 or A61F11/00 or subgroups thereof
  • A61F2250/0058—Additional features; Implant or prostheses properties not otherwise provided for
  • A61F2250/0067—Means for introducing or releasing pharmaceutical products into the body
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention relates to genetically engineered cells, and to the use thereof. Still more particularly, this invention relates to genetically engineered endothelial cells and the use thereof for expressing a therapeutic agent.
• the present invention is directed to genetically engineered endothelial cells, and the use thereof for expressing a heterologous protein.
• the heterologous protein is a therapeutic agen .
• endothelial cells which are transformed .with at least one gene which encodes for at least one heterologous protein, which, is preferably a therapeutic agent.
• a solid support which includesis-eiidothelial cells transformed with at least one gene which encodes for at least one heterologous protein, preferably a Jrherapeutic agent.
• the solid support is one which is compatible with blood and may, for example, be in the form of a blood vessel graft.
• endothelial cells which are transformed with at least one gene which encodes for at least one heterologous protein, preferably a therapeutic agent are implanted in a blood vessel.
• the endothelial cells employed in the present invention are endothelial cells derived from a mammal.
• the endothelial cells are obtained from a blood vessel.
• blood vessel as used herein includes veins, arteries and capillaries.
• the endothelial cells- which are genetically-, engineered include macrovascular and/or microvascular endothelial cells.
• the mammalian cells may be derived from a human or nonhuman mammal.
• the endothelial cells are preferably derived from a human.
• the endothelial cells are transformed with at least one gene which encodes for at least one heterologous protein which is preferably a therapeutic agent.
• the cells may be transformed in a manner in which the therapeutic agent is secreted from the transformed cells or may be transformed in a manner in which the therapeutic agent remains in or on the transformed cells.
• the mammalian endothelial cells are transformed with a suitable vector or expression vehicle which includes a gene for at least one therapeutic agent.
• the vector includes a promoter for expression in mammalian cells; for example, SV 40, LTR, metallothionein, PGK; CMV; ADA; TK; etc.
• the vector may also include a suitable signal sequence or sequences for secreting the therapeutic agent from the cells, * The selection " of a suitable promoter is deemed ' to be within the skill of the'art from the teachings herein.
• the expression vehicle or vector is preferably"a viral vector and in particular a retroviral vector.
• suitable viral vectors which can be modified to include a gene for a therapeutic agent, there may be mentioned: Harvey Sarcoma virus; ROUS Sarcoma virus, MPSV, Moloney murine leukemia virus, DNA viruses (adenovirus) etc.
• the expression vehicle may be in the form of a plasmid.
• the expression vehicle may also be in a form other than a vector; for example, transformation may be accomplished by liposome fusion, calcium phosphate or dextran sulfate transfection; electroporation, lipofection, tungsten particles etc.
• transformation may be accomplished by liposome fusion, calcium phosphate or dextran sulfate transfection; electroporation, lipofection, tungsten particles etc.
• the selection of a suitable vehicle for transformation is deemed to be within the scope of those skilled in the art from the teachings herein.
• a retroviral vector as the expression vehicle for transforming endothelial cells
• steps should be taken to eliminate and/or minimize the chances for replication of the virus.
• Various procedures are known in the art for providing helper cells which produce viral vector particles which are essentially free of replicating virus.
• Markowitz, et al. "A Safe Packaging Line for Gene Transfer: Separating Viral Genes on Two Different Plasmids.” Journal of Virology. Vol. 62, No. 4, pgs. 1120-1124 (April 1988); Watanabe, et al., "Construction of a Helper Cell Line for Avian Reticuloendotheliosis Virus Cloning Vectors, "Molecular and Cellular Biology. Vol. 3, No.
• the endothelial cells which are to be genetically engineered in accordance with the present invention may be derived from a mammal, and as hereinabove indicated, such endothelial cells may be obtained from an appropriate blood vessel, such as an artery, vein or capillary.
• an appropriate blood vessel such as an artery, vein or capillary.
• the procedure for obtaining such endothelial cells from the blood vessel of a mammal are generally known in the art and a representative procedure is disclosed in the Examples.
• endothelial cells genetically engineered ⁇ with a gene for a therapeutic agent; however, the scope of the invention is not to be limited thereby.
• the endothelial cells may be genetically engineered with a gene for a protein which is not a therapeutic agent; for example, a marker protein, such as beta-galactosidase.
• the endothelial cells are genetically engineered to include a gene for a therapeutic agent by the use of an appropriate vector, with the vector preferably being a retroviral vector.
• a representative procedure for genetically engineering endothelial cells by the use of a retroviral vector is described in the examples, and such general procedure and others may be employed for introducing other genes into mammalian endothelial cells.
• the procedure basically involves introduction of an appropriate promoter and DNA for the desired therapeutic agent into an appropriate retroviral vector.
• other material may be included in the vector such as a selection gene; for example a neomycin resistance gene; a sequence for enhancing expression, etc.
• The. ppropriate vector now containing,a gene foy at least one desired therapeutic, agent is employed for,.transducing mammalian endothelial cells by procedures generally available in the art.
• genetically engineered endothelial cells and in particular those genetically engineered with at least one gene for at least one therapeutic agent may be supported on a solid support.
• the solid support is preferably one which is.,biocompatible with blood whereby the solid support including the genetically engineered endothelial cells may be placed in communication with the blood system of a patient.
• the solid support may be employed in an extracorporeal device or implanted in a blood vessel (the term implant in a blood vessel includes a by-pass or a shunt for a blood, vessel) .
• the implantation may take the form of a blood vessel graft (the term graft includes a shunt or bypass). It is to be understood, however, that the solid support may take a variety of forms, such as pads, strips, gels, etc. and is not limited to grafts.
• the genetically engineered mammalian endothelial cells which include a gene for a therapeutic agent, may be implanted in a blood vessel of a mammal.
• the mammalian endothelial cells which are genetically engineered in accordance with the present invention are derived from a mammal, and the transformed endothelial cells are implanted in a blood vessel of a mammal of the same species.
• the genetically engineered mammalian endothelial cells are implanted in the blood vessel of a host from which the cells were originally derived; i.e., autologous cells are employed.
• endothelial cells are derived from a " blood vessel of a patient, genetically engineered to include a gene for at least one -therapeutic agent and the genetically engineered cells are implanted in a blood vessel of the patient from which they were* derived.
• autologous genetically engineered endothelial cells are employed for . in vivo production of a ' ⁇ therapeutic agent for treatment of a patient ⁇ i.e., gene therapy.
• the genetically engineered endothelial cells may be implanted in a blood vessel on a solid support or implanted directly onto a blood vessel (without the use of a solid support). It is also to be understood that the endothelial cells may be placed on a solid support in an extracorporeal device in communication with the blood system.
• the genetically engineered mammalian cells are implanted in a " blood vessel by providing a blood vessel graft which includes the genetically engineered endothelial cells.
• the graft now including genetically engineered endothelial cells may be inserted into a blood vessel of a host.
• a blood vessel graft which includes genetically engineered endothelial cells which are suitable for use in a mammalian host, which may be a human or nonhuman mammal.
• the blood vessel graft may be any one of a wide variety of vascular grafts and such grafts may be of various sizes.
• the graft may be used in a vein, an artery, or a capillary.
• the selection of appropriate grafts is deemed to be within the scope of those skilled in the art from the teachings herein.
• a synthetic vascular graft is preferred, it is possible within the spirit and scope of the present invention to provide a blood vessel derived from a host with genetically engineered endothelial cells and then graft such blood vessel back into the host.
• graft includes natural and synthetic grafts.
• the graft may be provided with genetically engineered endothelial cells in accordance with the present invention by seeding the genetically engineered endothelial cells onto a suitable blood vessel graft.
• Representative graft and procedure is disclosed in the Examples. The present invention is not limited to such 'grafts and procedures.
• Other' * grafts and procedures for seeding endothelial cells onto ' the graft are known in the art and may be used in the present invention.' For example, Herring et al Eds. Endothelial Seeding in Vascular Surgery (Grune & Stratton, Inc. Orlando 1987); Ziller et al Eds.
• polyesters for example DACRON
• expanded polytetrafluroethylene Gore-Tex
• polyurethanes coated polyurethanes; such as a silicone coated polyurethane manufactured by Corvita corporation in Miami, Florida
• tubular slotted stainless steel stents Johnson and Johnson which are coated with a substrate to permit adhesion of the endothelial cells to the stents; natural blood vessels, etc.
• the graft now including genetically engineered endothelial cells, may then be inserted into a blood vessel of a host.
• the procedures for placing a graft in an appropriate blood vessel are generally known in the art, and such procedures are applicable to the present invention.
• endothelial cells may be removed from a blood vessel of a patient, genetically engineered and returned to a blood vessel of the patient, without use of an impl.antable solid support.
• the endothelial cells which are genetically engineered with an appropriate therapeutic agent, may be genetically engineered in a manner such that the therapeutic agent is secreted into the blood, whereby such therapeutic agent may exert its effect upon cells and tissues either in the immediate vicinity or in more distal locations.
• the therapeutic agent may not be secreted from the cells., and exert its effect- within or on the genetically engineered endothelial cells upon substances that diffuse into the cell.
• adenosine deaminase memo may function "within the cell to inactivate ⁇ adenosine, a toxic metabolite that -accumulates in severe combined immunodeficiency syndrome;
• phenylalanine hydroxylase may function -within a cell to inactivate phenylalanine, a toxic metabolite in phenylketonuria, etc.
• the endothelial cells are transformed with a gene for at least one heterologous protein, preferably a therapeutic agent.
• a therapeutic agent is used in its hroadest sense and means any agent or material which has a beneficial effect on the host.
• the therapeutic agent may be in the form of one or more proteins.
• CD-4 CD-4; Factor VIII, Factor IX, von Willebrand Factor, TPA; urokinase; hirudin; the interferons ; tumor necrosis factor, the- interleukins , hematopoietic growth factors (G-CSF, GM-CSF, IL3 erythropoietin) , antibodies, glucocerebrosidase; ADA; phenylalanine hydroxylase, human growth hormone, insulin, etc.
• G-CSF hematopoietic growth factors
• ADA phenylalanine hydroxylase
• human growth hormone insulin
• endothelial cells In using the genetically engineered endothelial cells, it is possible to employ a mixture of endothelial cells which includes endothelial cells genetically engineered with a gene for a first therapeutic agent and endothelial cells genetically engineered with a gene for a second therapeutic agent. It is also possible to transform individual endothelial cells with more than one gene.
• the genetically engineered endothelial cells may be implanted in a blood vessel alone or in combination with other genetically engineered endothelial cells or with other genetically engineered cells, such as smooth muscle cells, fibroblasts, glial cells, keratinocytes, etc. *
• the genetically engineered endothelial cells may be employed to produce, in vivo, therapeutically effective amounts of a desired therapeutic agent for treating a patient.
• factors such as the half life of the therapeutic agent; volume of the vascular system; production rate of the therapeutic agent by the cells; and the desired dosage level are considered.
• the selection of such vectors and cells is dependent on the therapeutic agent and is deemed to be within the scope of those skilled in the art from the teachings herein.
• Figures la through Id depict a schematic illustration of vectors used in the present invention.
• Example 1 A To construct the pG2N retroviral vector of the drawing used to genetically engineer endothelial cells to produce rat growth hormone, an SV40 promoted neomycin resistance gene and a rat growth hormone cDNA were placed into the pB2 retroviral vector (Laboratory of Molecular Hematology, NIH). A growth hormone cDNA was obtained by digesting the plasmid RGH-1 (Nature 270, 494 (1977)) with Xho I and Mae III restriction endonucleases (Boehringer Mannheim Biochemicals) .
• This rat growth hormone cDNA was eletrophoretically isolated out of an agaorse gel and purified via binding/ lution to glass beads, Geneclean (BIO 101, LaJolla, California). This growth hormone cDNA was then blunted using the large fragment of DNA polymerase (Klenow) (New England Biolabs) and nucleotide triphosphates as recommended by the manufacturer. This fragment was then purified with Geneclean.
• the B2 vector was constructed in order to replace the Neo gene in N2, [M.A. Eglitis, P. Kantoff, E. Gilboa, W.F. Anderson, Science 230. 1395 (1985); D. Armentano et, al. , J. Virol. 61, 1647 (1987) and shown in the drawing] with a multiple cloning site.
• N2 was first digested with Eco RI, thereby releasing both the 5' and 3' LTRs'with the adjoining MoMLV flanking sequences.
• the 3' LTR fragment was ligated into the EcoRI site of the plasmid GEM4 (Promega Biotech).
• the 5' LTR fragment with its flanking gag sequence was then digested with Cla I, Hind III linkers were added, and the fragment was inserted into the Hind III site of pGEM4.
• the pB2 vector was digested with the Hindi restriction endonuclease (New England Biolabs), and phosphatased using calf alkaline phosphatase. (Boehringer Mannheim Biochemicals). The pB2 plasmid was then purified with Geneclean. The pB2 vector and the rat growth hormone cDNA were then ligated using T4 ligase (New England Biolabs). The ligation was then transformed into competent DH5 bacteria (Bethes da Research Labs). Colonies were then screened for a growth hormone cDNA containing vector. The new vector was called pG2.
• pG2 was then digested with BamHI (New England Biolabs), purified with Geneclean (Bio 101), and blunt ended with the Klenow fragment (New England Biolabs).
• a 340 base pair SV40 promoted neomycin resistance gene fragment was isolated from the pSV2CAT plasmid (ATCC accession number 37155) by digesting with PvuII and Hindlll (New England Biolabs). This fragment was isolated by agarose gel electrophoresis and purified with Geneclean.
• the SV40-neomycin resistance fragment was then ligated using T4 ligase (New England Biolabs) with pG2 and transformed into DH5 competent bacteria per the manufacturers instruction (BRL). Colonies were screened and the resulting plasmid construct was called pG2N.
• the SAX vector shown in the drawing was obtained as described in.-Proc. :NatL> Acad. -Sci. USA 8_3:6563 (1986).
• the recombinant vectors (N2,SAX, G2N) used in the study were each separately . transfected into the currently available retroviral vector packaging cell lines, including the amphotropic packaging lines, PA12 (Science ,225:630 (1984) and PA317 (Mol. Cell. Biol 6 ⁇ :2895 (1986), and the ecotropic line, Psi2 (Cell _3:153 (1983). These lines were developed in order to allow the production of helper virus-free retroviral vector particles .
• Aortic endothelial cells were obtained from New Zealand White rabbits (2-5 kilograms) by methods described previously for obtaining endothelial cells from bovine pulmonary artery (U.S. Ryan, M. Mortara, C. Whitaker, Tissue & Cell 12, 619 (1980)).
• the rabbit was anesthetized (1 ml sodium pentobarbital) and the aorta was removed and placed in Hanks buffered saline containing 3X antibiotics.
• the vessel was slit longitudinally and the luminal surface was scraped with a #11 scalpel blade taking care to scrape each area only once.
• the initial isolates were grown in Ryan Red medium [Ryan et al J. Tissue Cult. Methods. 10:3 (1986)], purified by selection of endothelial "islands" and passaged with a rubber policeman. Passaged cells were grown in
• a confluent 100 mm tissue culture dish (Costar) was harvested with a cell scraper. Following the dispersal of the cells by titurating 10-20 times with a 5 ml pipet, the cells were plated into 2-100 mm tissue culture dishes with 8 ml Ryan Red medium. After an overnight incubation, the medium was removed and 5 ml retroviral vector supernatant was added with Polybrene at a final concentration of 8 ug/ml. After a 2 hour incubation an additional 5 ml of Ryan Red was added to the dish. The cells were incubated overnight and the medium was replaced with 8 ml of Ryan Red. After another overnight incubation G418 was added to a final concentration of 200 ug/ml. The cells were the fed every 3-4 days with Ryan Red containing 200 ug/ml G418. The cell were subsequently maintained in Ryan Red without G418..
• G2N-infected RAEC that D had been selected in G418-**containing growth medium were harvested with a rubber policeman from 2 confluent T75 flasks and suspended in 5 ml of Ryan Red.
• The-cell suspension was titurated 6-7X with, a 6 cc syringe and, a 23 gauge needle.
• the cells were pelleted and resuspended in 1.25ml of Ryan Red.
• a vascular clamp was attached to one of a 10 cm X 4 mm (inner diameter) Corvita graft (Corvita Corp., Miami, FL) which is a silicone coated polyurethane graft.
• the cell suspension was vortexed and; introduced into the open end of the graft with a 3 cc syringe and 20 gauge needle.
• a second vascular clamp was attached to the open end and the graft was placed into a 50 ml conical tube filled with Ryan Red medium.
• the conical tube was capped) wrapped in parafilm, and placed into a roller bottle.
• the roller bottle was rotated overnight at 37 degrees Centr-igrade.
• the next day the clamps were removed and the graft was placed into a 500 ml bottle containing 150 ml of Ryan Red.
• the bottle was placed in an incubator equilibrated with 5% CO- , at 37 degrees C. It remained in the incubator and was periodically rotated for the next nine days.
• rGH continued to be secreted into the tissue culture medium at a rraattee ooff aapppprrooxxiimmaatteellyy 11000000 nngg//1100 cctells/day for at least 4 weeks after seeding the graft as follows
• the CD4 containing plasmid (pT4B, a gift of Richard Axel of College of Physicians and Surgeons Columbia University, New York, New York) was digested with the restriction endonucleases Eco RI and Bam HI New England Biolabs, Beverly MA) to release the CD4 gene which was isolated by agarose gel electrophoresis followed by purification via binding/elution to glass beads (using the geneclean product, BIO 101,*.. La Jolla CA in the manner recommended by the manufacturer).
• the CD4 fragment was ligated (using T4 DNA ligase as recommended by the supplier, New England Biolabs) into Eco RI plus Bam HI cut Bluescript cloning vector (stratagene Co. La Jolla CA) .
• the ligation was then transformed into competent DH5 alpha bacteria (Bethesda Research Labs, Gaithersburg MD) and white colonies were isolated and screened for proper insert size to yield the plasmid pCDW.
• the plasmid SV2neo obtained form American Type Culture Collection, Rockville MD
• a synthetic polylinker sequence from the pUC-13 vector (Pharamicia, Piscataway NJ) was inserted (via T4 DNA ligase) in place of the neo gene of pSV2neo.
• This ligation was transformed into DH5 bacteria (Bethesda Research Labs) and colonies screened for the presence of restriction enzyme sites unique to the polylinker to yield the vector pSVPL.
• the pSVPL expression vector was further modified by the insertion of an Xho I linker (conditions and reagents supplied by, New England Biolabs) into the Pvu II site on the 5' side of the SV40 early region promoter to produce pSVPLX.
• the pCDW and pSVPLX plasmids were digested with enzymes Hind 3 plus Xba I, (New England Biolabs) and their DNAs isolated (using the Gene Clean product) following agarose gel electrophoresis.
• oligonucleotide adaptor to produce a mutant form of the CD4 gene.
• This adaptor has the unique property that when inserted into the Nhe I site of the CD4 gene it produces the precise premature termination of the CD4 protein amino acid sequence while regenerating the Nhe I site and creating a new Hpa I site.
• This oligonucleotide adaptor (synthesized by Midland certified reagent Co.) was produced by annealing two phosphorylated oligonucleotides; 1) 5' CTAGCITGAGTGAGIT 3', 2) AACTCACTCAAG and then this product was ligated into the site of pSVCDW.
• the ligation reaction was then cleaved with Hpa I and then Xho I linkers were added (New England Biolabs).
• the linker reaction was terminated by heating at 65C for 15 min. and then subjected to digestion with Xho I restriction endonuclease (New England Biolabs).
• This reaction was then subjected to agarose gel electrophoresis and the fragment containing the SV40-CD, adaptor isolated (Geneclean).
• the retroviral vector N2 was prepared to accept the SV40-CD,-adaptor fragment by digestion with Xho I and treatment with Calf intestinal phosphatase (Boehringer Mannheim, Indianpolis IN).
• CD4 expression cassette was performed with an insert to vector ratio of 5:1 and then transformed into DH5 competent bacteria (Bethesda Research Labs). Constructs were analyzed by restriction endonuclease digestion to screen for orientation and then grown up in large scale.
• SSC The construct where the SV40 virus promoter is in the same orientation as the viral LTR promoters is known as SSC while the construction in the reverse or reverse orientation is called SCSC.
• the SSC vector is packaged into PA317 cell line as described by Miller et al supra, to provide PA 31-7 cells • capable of producing soluble CD4 protein.
• the SSC vector packaged PA 317 cells were used to transduce rabbit endothelial cells as hereinabove described.
• the transduced endothelial cells were found to express soluble CD-4.
• ECGF (crude brain extract ) 7.2mg
• Selected rat endothelial cells were transduced with the N-7, SAX, G2N and SSC vectors by the following procedure:
• tissue culture media for 16 hrs . at 37°C (5% CO-).
• tissue culture media for 16 hrs . at 37°C (5% CO-).
• -Aspirate off media virus containing
• G418 800ug/ml
• culture media -Select for one to two weeks changing media every two days.
• rat endothelial cells transduced with G2N expressed rat growth hormone in vitro as follows:
• Example 2 Endothelial cells were harvested from segments of adult sheep jugular vein, carotid artery, and femoral vein using the method of Jaffee et al. J.Clin.Invest.. 52:2745-2756 (1973). A total of four vessels from three sheep were used. Identification of harvested endothelial cells was confirmed by their cobblestone structure and by confirmed by their cobblestone structure and by binding of the fluorescent ligand Dil-Acetyl-LDL J.Cell.Biol. 99:2034-2040 (1984)). (Biomedical Technologies, Stoughton, Massachusetts).
• a murine ecotropic packaging line capable of transmitting the ⁇ -galactosidase-containing "BAG" vector was, provided by Constance Cepko (Harvard University, Cambridge, Massachusetts). Supernatant from this packaging line was used to generate an amphotropic packaging line from PA-317 cells.
• a human t-PA cDNA in plasmid pPA34'f) (J.Biol. Chem. 260 t:11223-11230(1985)) was provided by Sandra Degan (University of Cincinnati, Cincinnati, Ohio).
• This t-PA cDNA was used, through several subcloning steps, to construct a t-PA containing retroviral vector, B2NSt analogous in construction to the SAX vector.
• the corresponding plasmid based on the B2 plasmid, (Science). 343:220-222 (1989)) was transfected into G .
• PE-86 cells J.Virol.. 62:1120-1124 (1988)
• supernatant from these cells thereby, generating amphotropic packaging clones capable of transmitting the t-PA gene.
• Endothelial cells were transduced by incubation for 2 hours with supernatant-containing virions with the retroviral vector, along with 8 ⁇ g/ml G-418 for at least 16 days.
• Duplicate cultures of cells from each vessel harvest were transduced simultaneously with either the t-PA- or ⁇ -galactosidase-containing retroviral vector and, then, cultured, passaged, and selected using identical procedures. In this manner, the t-PA- and ⁇ -galactosidase-transduced cells served as controls for one another in experiments involving either ⁇ -galactosidase activity or t-PA secretion.
• Coverage of the stent surfaces was monitored both by phase-contrast microscopy and by incubation of the stents for 4 hours in medium containing Dil-Acetyl-LDL followed by fluorescence microscopy.
• Supernatant to be assayed was collected above confluent monolayers in 35-mm dishes, 48 hours after addition of 2 ml fresh medium.
• the stent was transferred to a new well containing fresh medium and, then, began a timed collection of culture medium.
• Harvested supernatant was centrifuged at 15,000g for 15 minutes to remove cellular debris, made 0.01% with Tween-80, and frozen at -70°C until assayed.
• the rate of t-PA secretion in nanograms per 10 cells per 24 hours was calculated using a confluent cell density of 3 x 10 cells per cm 2 of tissue culture ⁇ plastic (data not shown) .
• E. Seeded stents were incubated in medium containing Dil-Acetyl-LDL for 4 hours before expansion.
• the stents were visualized by fluorescence microscopy to confirm endothelial coverage, and, then, manually placed over a deflated 3.0-mm diameter coronary angioplasty balloon catheter (Scimed Life Systems, Maple Grove, Minnesota). After balloon inflation to 4-6 atmospheres, resulting in complete stent expansion, the balloon was deflated and the stents were removed from the catheters and, again, viewed by fluorescence microscopy.
• H Endothelial cells from all four vessels, when transduced with the t-PA vector, secreted immunoreactive t-PA.
• Rates of t-PA secretion (mean ⁇ SD of duplicate tissue culture wells, expressed as ng/10 cells/24 hours) were femoral vein, 370 ⁇ 8; carotid artery, 660 ⁇ 240; jugular vein 1, 230 ⁇ 6; jugular vein 2, 200 ⁇ 18.
• t-PA production by the ⁇ -galactosidase,- transduced cells was below the lower limit of sensitivity of the assay (i.e., less than 5 ng/10 cells/24 hours) in all of the supernatants tested.
• t-PA secretion by the ⁇ -galactosidase-transduced cells on each of three stents was below the limit of detection of the assay.
• the measured t-PA secretion from each of three lines of transduced cells was used both before and after they were seeded onto stents to calculate the surface area of the stents. This calculation is based on the assumption that the density of the cells and " the rate of t-PA secretion do not change when the cells are on the stents.
• a stent surface area (mean ⁇ SD) of 48 ⁇ 19 mm was calculated, not significantly different from the manufacturer's value of 42 mm 2 (personal communication, Johnson and Johnson Interventional Systems, Warren, New Jersey).
• an intravascular stent seeded with endothelial cells as hereinabove described may produce a local thrombolytic environment in vivo.
• High level secretion of t-PA adjacent to a forming clot may permit t-PA to be concentrated through the high affinity binding of t-PA to fibrin. (Thorsen, et al., Thromb P. Haemorrh, 28:65-74 (1972)).
• fibrinolytic activity would be directed to microthrombi beginning to form on the stent surface or downstream, thus preventing the formation of occlusive thrombi.

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